JP2983223B2 - How to keep microorganisms alive - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the use of seaweed for acid, alkali, heating, freezing,
The present invention relates to a method for protecting microorganisms and maintaining viable bacteria under special environmental conditions such as drying.

[Conventional technology]

2. Description of the Related Art In recent years, attempts to effectively utilize the functions of microorganisms have been actively made in various industrial fields such as foods. In general, when using microorganisms in the food industry, etc., it is known that the microorganisms exert their functions in a living state and utilize their functions in a dead bacterial state in order to utilize the metabolic ability of the microorganisms. . In addition, in the case of foods containing useful microorganisms and the like, the viability of microorganisms in the product often determines the value of the product. Therefore, it is important to maintain the viable state of the microorganism in the manufacturing process or in the product in order to fully utilize the function of the microorganism in industry. However, in the manufacturing process of foods and the like, generally, there are many environments that are not favorable for survival of microorganisms such as temporary pH change, freezing, and high temperature. If you have a manufacturing process that is not preferred for the survival of microorganisms,
(1) A microorganism having a desired function cannot be used. (2) It is considered that the function of microorganisms cannot be fully utilized, and (3) complicated steps are required. This is a major problem in the industry that uses microorganisms.

For example, in the food industry, many foods utilizing lactic acid bacteria represented by yogurt are commercially available. recent years,
With the advance of science, it has become clear that these lactic acid bacteria work effectively in the intestine, and they are receiving attention. However, in products using these lactic acid bacteria, the survival number of the lactic acid bacteria is expected to decrease during storage because the pH in the product is generally low.
It is also conceivable that gastric acid, which has a low pH when passing through the stomach after oral ingestion, kills it. However, at present, many products utilizing these lactic acid bacteria do not take into account the life and death of the lactic acid bacteria in the product or in the gastrointestinal tract after oral ingestion. Naturally, useful intestinal bacteria are considered to act more effectively in the viable state, and if the viable state is maintained in food or after oral ingestion, the range of utilization of lactic acid bacteria will be greatly expanded.

On the other hand, in the bread making industry, there are also problems such as that yeast is killed during the freezing of bread dough, sufficient fermentation is not performed, and bread does not expand. Furthermore, many lyophilized preparations of useful intestinal bacteria such as bifidobacteria for the purpose of improving the intestinal flora are commercially available, but these lyophilized preparations generally have a certain limit in the survival rate. Was.

Conventionally, as a method for allowing microorganisms to survive in a special environment in which microorganisms are difficult to survive, attempts have been made to improve strains and to isolate new strains that are resistant to external environments. Also,
For dairy products containing useful lactic acid bacteria such as yogurt, an additive (JP-A-62-11053) has been proposed which is considered to be effective in increasing the acid resistance of the lactic acid bacteria in the product and increasing the survival rate. On the other hand, protective agents used in the freezing method, which is one of the microorganism preservation techniques, such as glycerin, methyl sulfoxide, skim milk powder, or glucose, sucrose, lactose, raffinose, and other carbohydrates can also increase the survival rate of microorganisms in frozen foods. It can also be applied as

However, the additives used in yogurt only consider viability in yogurt, so it is difficult to maintain a viable bacterial state in gastric juice after oral ingestion at a lower pH, and low pH In foods and the like, the effect of addition itself cannot be expected.

Furthermore, even when a cryoprotectant is applied, it cannot be added to food because there is no protective agent that can be added to food, and even if it can be added, the flavor may change. At present it is difficult to apply to food.

[Problems to be solved by the invention]

The present invention has been made in order to solve the above-mentioned problems of the prior art, and the present invention protects microorganisms in an environment where microorganisms are difficult to survive, such as acidity, alkalinity, heating, freezing, and drying. It is an object to provide a method for maintaining a viable bacterial state.

Further, the present invention provides a method for protecting microorganisms, which is inexpensive and can be easily applied to foods, medicines, etc. without requiring special techniques, and which can expand the range of use of microorganisms and the effect of using microorganisms more and more. The purpose is to do. Then, as a result of searching for substances that protect microorganisms more widely in nature, they found that seaweed powder or an extract thereof had a microorganism-protecting action, and completed the present invention.

[Means for solving the problem]

That is, the present invention is characterized in that by contacting the microorganisms with seaweed powder or an extract thereof, the microorganisms are kept alive in an external environment such as an acidic region, an alkaline region, a high temperature region, a low temperature region, and a dry region. Is what you do.

Hereinafter, the method of the present invention will be described more specifically. Specific examples of the seaweed used in the present invention include seaweeds of the genus Laminaria, the genus Ascophilum, the genus Wakame, and the genus Aonori. Seaweed is used by contacting microorganisms in the form of a dry powder or an extract thereof. Furthermore, a higher protective effect can be obtained by fractionating the seaweed into the epidermis and the core and using the fractionated core in the form of a dry powder or an extract thereof. The core portion is white and almost transparent, tasteless and odorless after swelling, so that it is particularly suitable for application to foods.

Seaweed powder, dried seaweed 150 ~ 60mesh using a crusher
Prepared by grinding until passing through a sieve. When fractionating the core part of the seaweed, use the dried seaweed in distilled water or tap water, and re-water it for at least 12 hours. The drawn core is sufficiently dried and then crushed by the above-mentioned method to prepare a core seaweed powder.

In the present invention, the seaweed powder prepared and prepared as described above is used as an active ingredient, and the culture solution containing a viable microbe of a useful microorganism is added in an amount of 0.
1% to 5.0% by weight, preferably 0.5% to 3.0% by weight
Used in addition.

Furthermore, a substance for maintaining the survival of microorganisms can be obtained by extracting seaweed or its core with distilled water or tap water. And
This substance for maintaining the survival of microorganisms is used as an active ingredient having higher activity.

Examples of microorganisms to which the method of the present invention can be applied include lactic acid bacteria exemplified by Lactobacillus, Streptococcus, Bifidobacterium, Leuconostoc, Pediococcus, etc., Saccharomyces, Schizosaccharomyces, Candida. And the like. However, applicable microorganisms are not limited to these. Conventionally, the method of the present invention is applied to microorganisms whose survival rate is significantly reduced by treatments such as acid, alkali, heating, freezing, and drying. By doing so, it is possible to maintain a high survival rate over a long period of time.

The survival rate of the microorganisms described above can be maintained in a special environment where it is conventionally difficult for the microorganisms to survive, for example, in an acidic region, an alkaline region, a high temperature region, a low temperature region, and a dry region. Particularly in the acidic region of pH 0.5 to 5.0, pH 7.5
~ 12.0 alkaline range, 60 ° C ~ 100 ° C high temperature range, -8
The effect is remarkable in a low temperature range of 5 ° C to 0 ° C.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

In addition, the measurement of the survival rate under various environments when the above-mentioned active ingredient was brought into contact with microorganisms was in accordance with the test method described below.

Test method An appropriate amount of the seaweed powder prepared as described above is added to a culture obtained by culturing a microorganism to be used in a standard medium of the microorganism until a stationary phase, and at an optimal culture temperature of the strain to be used for 1 hour or more. Contact the bacterial cells with the seaweed powder. The mixture of the seaweed and the cells is aseptically collected by centrifugation. A fixed amount of the obtained seaweed and the seaweed-contacting cells are immersed in a hydrochloric acid solution having a pH of 5.0 or less to perform an acid sterilization treatment. Cells are taken out at regular intervals after being immersed in a hydrochloric acid solution, inoculated on a plate medium, and the viable cell count is measured. In addition, the hydrochloric acid solution
The pH is measured after immersing the cells and seaweed.

Example 1 Lactobacillus acidophilus (Lactobacillusaci
dophilus IAM 12475) in a medium for general lactic acid bacteria (composition is No. 1)
To a culture obtained by inoculation of Table 2) and culturing for 24 hours, 0.3% by weight of seaweed powder of the genus Wakame at a final concentration was added,
Contact the microbial cells with the seaweed powder for more than an hour. The mixture of the seaweed and the cells was aseptically collected by centrifugation.

Table 1 Composition of general lactic acid bacteria medium Glucose 11.0 g Yeast extract 5.5 Polypeptone 12.5 Sodium acetate (CH ₃ COONa) 1.0 Potassium phosphate (KH ₂ PO ₄ ) 250.0 mg Dipotassium phosphate (K ₂ HPO ₄ ) 250.0 Magnesium sulfate (MgSO ₄ ) ₄ · 7H ₂ O 100.0 ferrous sulfate _{(FeSO 4 · 7H 2 O)} 5.0 manganese sulfate _{(MnSO 4 · 4-6H 2 O)} 5.0 ( agarose 15.0 g) · · plait medium distilled water 1000.0M (initial pH) 6.8 A fixed amount of the obtained seaweed and seaweed-contacting cells were immersed in a hydrochloric acid solution of pH 1.0 to perform acid sterilization, and after immersion in the hydrochloric acid solution, the cells were taken out at intervals of 20 minutes and inoculated into a plate medium. The results are shown in Table 2.

As is clear from this table, the cells which were brought into contact with the active ingredient according to the present invention, as compared with the non-contact cells as a control,
The viable cell count of lactic acid bacteria after 60 minutes is 2.5 × 10 ³ cells / m,
The effect was remarkable.

Example 2 2.5 g of distilled water was added to 0.5 g of seaweed powder of the genus Laminaria and sterilized in an autoclave (at 115 ° C. for 20 minutes). On the other hand, Lactobacillus bulgaricus (Lacto-bacillus bulgaricus I)
AM 1120), and cultured for 24 hours according to a conventional method to obtain a culture solution having a cell count of 1.6 × 10 ¹⁰ cells / m. 1.0% (by weight) of the above-mentioned sterilized seaweed powder was added to the culture solution, stirred with a mixer, and left at 37 ° C. for 3 hours. After standing, the seaweed and the seaweed-contacting cells were collected by centrifugation, suspended in a pH 1.4 HCl solution so that the number of cells was 1.6 × 10 ¹⁰ cells / m, and subjected to acid sterilization. Cells were taken out of the HCl solution every 10 minutes, and the viable cell count was measured. 90 after immersion in HCl solution
One minute later, the viable cell count of Lactobacillus bulgaricus contacted with seaweed was 1.4 × 10 ⁵ cells / m (Table 3). In addition, in the control to which the seaweed powder was not added, the survival of the bacteria could not be confirmed 90 minutes after the immersion.

Example 3 5 g of dried seaweed of the genus Ascophyllum
It was immersed in 1 liter of distilled water and moistened at 5 ° C. for 24 hours. The sufficiently wet seaweed was fractionated into a green epidermis and a white core using a cutter knife. After fractionation, the epidermis and the core were dried in an oven at 60 ° C. for 6 hours. Each dried product was ground in a sample mill until it passed through a 115 mesh sieve. 35 g and 45 g of dry powder were obtained for the epidermis and the core, respectively. After sterilizing the core powder according to the procedure described in Example 1, 3.0% of the culture solution prepared according to the procedure described in Example 1 was used.
(Weight) was added, and the cells (Lactobacillus bulgarcas) were brought into contact with seaweeds in the same manner as in Example 1. Next, the cells and seaweed were recovered according to Example 1, and the cells were diluted and immersed in an HCl solution having pH 1.1, 1.3, and 1.5 so that the number of cells was 10 ⁶ / m. Cells were taken out of the HCl solution every 5 minutes after immersion and inoculated to a general lactic acid bacterium medium so that OD660 = 0.01. 37 ℃
The growth of the cells after culturing for 48 hours was measured to confirm the survival of the cells.
When immersed in a pH 1.1 HCl solution for 30 minutes, the seaweed-contacting bacterial cells grew sufficiently after being cultured in a general lactic acid bacteria medium for 48 hours, and their survival was confirmed (FIG. 1). In the control to which the seaweed powder was not added, the cells immersed in the HCl solution for 5 minutes or more could not be confirmed to grow on the general lactic acid bacteria medium. In addition, when the seaweed was used without fractionation, the protective effect was lower than when only the core was used.

Example 4 Saccharomyces cerevi
siae IAM 4274) was inoculated into a yeast medium (YM medium) and cultured at 30 ° C. for 24 hours according to a conventional method to obtain a culture solution containing 10 ⁹ cells / m viable bacteria. The cells recovered by centrifugation from 3 m of the culture solution were suspended in 3 m of sterile physiological saline to prepare a cell suspension. 30 mg of the sterilized seaweed powder prepared by the method described above was added to the cell suspension, mixed well, and then frozen in a freezer at −25 ° C. for 12 hours. After 12 hours, a melting treatment was performed for 10 minutes in a thermostat at 30 ° C., and then the number of viable cells was measured. As a control, yeast that had been frozen without adding ground seaweed was used. The results are shown in Table 4. In a system to which ground seaweed is added,
The number of viable cells after freezing and thawing was almost unchanged from that before freezing, and the viable cells were maintained. On the other hand, in the control-free system, the number of viable cells after freezing and thawing was reduced to 1/20 that before freezing, and the microbial protection of seaweed against freezing was remarkable.

Table 4 Reduction of viable yeast cell count by freezing and thawing Viable cell count before freezing and viable cell count after freezing and thawing Seaweed non-contact cells 1.03 × 10 ⁹ 5.0 × 10 ⁷ Seaweed contact cells 1.03 × 10 ⁹ 1.0 × 10 ⁹ [Effects of the Invention] As described above, the present invention can protect microorganisms and maintain a viable state in an environment such as acidity or freezing. According to the present invention, even in an environment where it was conventionally considered difficult to maintain microorganisms in a viable state,
Microorganisms can survive, application to foods, medicines, etc. can be facilitated, and the range of use of microorganisms and the effect of using microorganisms can be further increased.

[Brief description of the drawings]

FIG. 1 is a graph showing the growth amount of bacterial cells in Example 3.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/20 C12R 1: 225) (56) References JP-A-61-231992 (JP, A)

Claims (13)

(57) [Claims] 1. A microorganism other than basidiomycete is contacted with a seaweed powder or an extract thereof to protect the microorganism in an external environment such as an acidic region, a high temperature region, a low temperature region or a dry region where the microorganism is difficult to survive. How to stay alive. 2. The seaweed is of the genus Laminaria, the genus Ascophyllum,
The method according to claim 1, wherein the method is of the genus Wakame or Aonori. 3. The method according to claim 1, wherein the microorganism is a lactic acid bacterium. 4. The method according to claim 3, wherein the lactic acid bacterium is a microorganism belonging to the genus Lactobacillus, Streptococcus, Bifidobacterium, Leuconostoc or Pedococcus. 5. The method according to claim 1, wherein the microorganism is yeast. 6. The yeast according to claim 5, wherein the yeast belongs to the genus Saccharomyces, Schizosaccharomyces or Candida.
The method described in. 7. The method according to claim 1, wherein the external environment of the acidic region is in the acidic range of pH 0.5 to 5.0. 8. The method according to claim 1, wherein the external environment in the high temperature region is a heating temperature range of 60 ° C. to 100 ° C. 9. The method according to claim 1, wherein the external environment in the low temperature range is a freezing temperature range of -85 ° C. to 0 ° C. 10. The method of claim 1, wherein the external environment of the drying area is dry. 11. The method according to claim 1, wherein the microorganism is a microorganism living in a food. 12. The method according to claim 1, wherein the seaweed powder is obtained by pulverizing dried seaweed or fractionating the seaweed into an epidermis and a core, and drying and pulverizing the core. 13. An external environment of an acidic region, a high-temperature region, a low-temperature region or a dry region in which microorganisms other than basidiomycetes are difficult to survive, characterized by containing a seaweed or a dry powder or extract of a core thereof. A substance for protecting and keeping the microorganism alive therein.